I'm trying my hand at tissue culture for the first time today. I'm going to start with seeds. I'll probably try some plant tissue also, but this time of year choosing good tissue is tough because my plants are mostly dormant, so there isn't much new growth. Choosing new growth to work with is important because the older the tissue, the more overrun with bacteria it is, which means it's harder to sterilize. Let me see if I can answer your questions:
1. What are the optimum conditions for success?
This is a hard question to address because there are so many conditions, but the basic answer is sterile media and sterile tissue (seeds, flower stalk or plant tissue).
2. What is used to ensure that the tissue and growing container are sterilised?
To sterilize the growing container and media most people that do TC at home use a pressure cooker. I have an 8 quart pressure cooker. Most pressure cookers are rated at 15PSI, which is ideal for sterilization. You fill the growing containers (most people use baby food jars) with the media and sterilize at 15PSI for 20 to 30 minutes (time depends on the volume of media in each container and what elevation you're at).
Sterilizing the tissue is the challenging part. For Dionaea, seeds are the easiest to sterilize, followed by flower stalk, followed by cuttings. The sterilization techniques vary, but here's what I'm going to try today:
1) 10% bleach for 15 minutes
2) Rinse in sterile water
3) Transfer to media
1) Dip the cutting in the 90% alcohol quickly. Hold it above the alcohol to let any excess drain.
2) Drop the cutting in 3% peroxide for 2 minutes
3) Drop the cutting in 10% bleach mix for 15 minutes
4) Drop the cutting in sterile water for 10 minutes
5) Drop the cutting in 2% PPM (plant preservative mixture) solution for 30 to 60 minutes
6) Transfer to media
3. What is the growing media (agar I'm guessing) and what is added to it to help the tissue grow?
I think this is a common misconception. Agar is inert and doesn't provide anything to the media other than to help it solidify a bit so that the seeds or tissue isn't floating around. The media for carnivorous plants is typically 25% to 50% (I'm going to try 50%) concentration of Murashige & Skoog (MS) basal salt mixture. To stimulate root growth, Auxins are used. And to stimulate cell growth, Cytokinins are used. For an Auxin, I think most people use IBA and for a Cytokinin, BAP is commonly used. I've read varying ratios, but 5ml BAP to 0.25ml IBA per liter of media is what one person that TCs Dionaea told me he uses. The TC kit that I have recommends 3ml of BAP per liter of media. From what I've read, for Dionaea you don't even really need it. I think I'm going to try some with and some without it. The other thing that is added to the media is sugar. Regular table sugar is fine and it should be 2 tablespoons (30g) per liter of media.
4. Can it be done at home or do I need specialist equipment or a lab?
It can be done at home. I ordered my kit from here:http://www.kitchenculturekit.com/index.htm
It comes with a manual and it is a very good place to start. I'd love it if someone else on this forum got into TC and we could exchange notes. It sounds like sterilizing tissue is a trial and error process and I can't seem to get many people that have been successful to share their technique with me. So, it would be very valuable to have another person working on TC at the same time so that we could exchange notes.
Leave the meat for our pet plants